neurodegenerative disease individuals with progranulin ...william w. seeley,3 bruce l. miller,3...

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NEURODEGENERAT IVE D I SEASE

1Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco,CA 94158 USA. 2National Institute of Neurological Disorders and Stroke, NationalInstitutes of Health, Bethesda, MD 20892, USA. 3Department of Neurology, Uni-versity of California, San Francisco, San Francisco, CA 94158 USA. 4Department ofPathology, University of California, San Francisco, San Francisco, CA 94158, USA.5Department of Pathology, National Taiwan University Hospital, Taipei City,Taiwan. 6INSERM UMR 913, Université de Nantes, Nantes, France. 7Departmentof Neurology, Medical Faculty, Heinrich-Heine University Duesseldorf, Duesseldorf,Germany. 8Department of Ophthalmology, University of California, San Francisco,San Francisco, CA 94158, USA. 9Department of Pathology, Northwestern UniversityFeinberg School of Medicine, Chicago, IL 60611, USA. 10Department of Pathology,Stanford Medical School, Palo Alto, CA 94305, USA. 11Department of Pathology andLaboratory Medicine, University of British Columbia, Vancouver, Canada. 12Center forHuman Genetic Research, Massachusetts General Hospital, Harvard Medical School,Boston, MA 02114, USA. 13Pathology Service, Veterans Affairs Medical Center, SanFrancisco, CA 94141, USA.*Corresponding author. Email: [email protected] (L.G.); [email protected](A.J.G.)

Ward et al., Sci. Transl. Med. 9, eaah5642 (2017) 12 April 2017

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Individuals with progranulin haploinsufficiency exhibitfeatures of neuronal ceroid lipofuscinosisMichael E. Ward,1,2,3 Robert Chen,1,3 Hsin-Yi Huang,4,5 Connor Ludwig,1,3 Maria Telpoukhovskaia,1

Ali Taubes,1 Helene Boudin,4,6 Sakura S. Minami,1 Meredith Reichert,1 Philipp Albrecht,7

Jeffrey M. Gelfand,3 Andres Cruz-Herranz,3 Christian Cordano,3 Marcel V. Alavi,8 Shannon Leslie,1

William W. Seeley,3 Bruce L. Miller,3 Eileen Bigio,9 Marek-Marsel Mesulam,9 Matthew S. Bogyo,10

Ian R. Mackenzie,11 John F. Staropoli,12 Susan L. Cotman,12 Eric J. Huang,4,13

Li Gan,1,3* Ari J. Green3,8*

Heterozygous mutations in the GRN gene lead to progranulin (PGRN) haploinsufficiency and cause frontotemporaldementia (FTD), a neurodegenerative syndrome of older adults. HomozygousGRNmutations, on the other hand, leadto complete PGRN loss and cause neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease usually seen inchildren. Given that the predominant clinical and pathological features of FTD and NCL are distinct, it is controversialwhether the diseasemechanisms associated with complete and partial PGRN loss are similar or distinct. We show thatPGRN haploinsufficiency leads to NCL-like features in humans, some occurring before dementia onset. Noninvasiveretinal imaging revealed preclinical retinal lipofuscinosis in heterozygousGRNmutation carriers. Increased lipofuscinosisand intracellular NCL-like storage material also occurred in postmortem cortex of heterozygous GRNmutation carriers.Lymphoblasts fromheterozygousGRNmutation carriers accumulated prominent NCL-like storagematerial, which couldbe rescued by normalizing PGRN expression. Fibroblasts from heterozygous GRN mutation carriers showed impairedlysosomal protease activity. Our findings indicate that progranulin haploinsufficiency caused accumulation of NCL-likestoragematerial and early retinal abnormalities in humans and implicate lysosomal dysfunction as a central disease pro-cess in GRN-associated FTD and GRN-associated NCL.

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INTRODUCTIONNeurodegenerative diseases of adulthood are usually thought to occurviamechanisms distinct from those of childhood. Neuronal ceroid lipo-fuscinosis (NCL) is the most common neurodegenerative disease ofchildhood. Progranulin-related frontotemporal dementia (FTD), a neuro-degenerative syndrome of older adults, is the most common cause ofdementia in those <60 years old. Individuals affected by FTD usuallypresentwith behavioral changes, language difficulties, ormotor symptoms(1).Heterozygousmutations in the progranulin (GRN) gene are commoncauses of familial FTD. Such mutations lead to progranulin (PGRN)haploinsufficiency, reducing PGRN expression by 60 to 70% via nonsense-mediatedmRNAdecay (2). PGRNhasbeen reported to regulatenumerouscellular processes, including cell survival, inflammation, and proteinclearance (3–12). However, it is not understoodwhy partial loss of PGRNcauses neurodegeneration.

Recently, we discovered that retinal degeneration occurs in preclinicalheterozygousGRNmutation carriers (13), a phenotype also observed in

Grn knockout mice (13, 14).Grn knockout mice also develop prominentdeposits of autofluorescent aggregates known as lipofuscin throughoutthe central nervous system (CNS) (7, 15, 16). Ultrastructurally, this storagematerial bears a striking resemblance to storage material found in NCL, adisease clinically characterized by early vision loss and retinal atrophy. In-dividuals with homozygous GRN mutations develop a clinical syndromeconsistent with NCL (vision loss followed by seizures and ataxia) (17–19).Electron microscopy (EM) analysis of lymphocytes from one of thesesubjects revealed NCL-like storage material (18).

Classically, NCL encompasses a family of related genetic syndromeswith shared clinical and pathologic features, which together are themajorcause of neurodegeneration in childhood. Vision loss is often an earlyclinical feature ofNCLand canprecede development of other neurologicalsymptoms,which usually include seizures, ataxia, cognitive dysfunction,and early death (20).Most of the knownmutations associatedwithNCLcause loss of function in genes necessary for normal lysosome function.In keepingwith this, deposition of autofluorescent intralysosomal storagematerial known as lipofuscin is a pathological hallmark of NCL (21).

Although it is generally agreed that NCL can be caused by completeloss of PGRN, whether FTD caused by partial loss of PGRN shares sim-ilar clinical, pathological, ormechanistic featureswithNCL remains con-troversial. Unlike theGrn knockoutmice, heterozygousGrnmice do notdevelop CNS lipofuscinosis (7). Furthermore, the classic clinical andpathological findings considered characteristic of GRN-associated FTDappear distinct from those inNCL (namely, early behavioral/language ab-normalitieswith pathologic TDP-43mislocalization in FTDversus visionloss, seizures, and ataxiawith pathologic lipofuscin accumulation inNCL).However, a comprehensive evaluation of humanswith PGRNhaploinsuf-ficiency for clinicopathological features of NCL has not been conducted.

On the basis of our previous observations of early retinal degenera-tion in humans with heterozygous GRN mutations—which could beconsistent with an NCL phenotype—we suspected that other NCL-like

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featuresmay occur in these patients but may have been overlooked.Weexamined retinas of living presymptomatic heterozygous GRN muta-tion carriers by noninvasive autofluorescence imaging and analyzed post-mortem cortical tissues from FTD subjects with heterozygous GRNmutations. In addition, we imaged lymphoblasts from heterozygousGRN mutation carriers and their normal siblings by EM to quantifyNCL-like storage material. We enhanced PGRN expression in lympho-cytes from heterozygous GRNmutation carriers to assess the effects ofPGRN onNCL-like structures and storage material. Finally, we directlyassessed lysosomal function in fibroblasts from heterozygousGRNmuta-tion carriers. Our findings indicate that core clinicopathological featuresof NCL occur in humans with PGRN haploinsufficiency caused by het-erozygous GRN mutations, implicating lysosomal dysfunction as apotential disease process in GRN-associated FTD.

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RESULTSLipofuscinosis and NCL-like storage material in retinalneurons from Grn knockout miceVision loss is often the first symptom noticed by patients with someforms of NCL, and it is accompanied by retinal atrophy and depositionof autofluorescent lipofuscin. We reported retinal degeneration accom-panied by electrophysiological dysfunction in Grn knockout mice (13),which was subsequently reported by others (14). Similar to findings incortical neurons from Grn knockout mice and in various NCL animalmodels (7), we observed NCL-like lysosomal storage material in retinalneurons from Grn knockout mice (Fig. 1A).

Age-dependent autofluorescent lipofuscin deposition was observedthroughout the retina in Grn knockout mice, reminiscent of autofluo-rescent lipofuscin deposits in NCL (Fig. 1, B and C). In addition, sub-retinaldrusen-like autofluorescentdepositswerehigher inagedGrnknockoutmice than in control animals: 16.0 + 2.7 versus 3.2 ± 1.9 deposits perretinal cross section (n = 5, P < 0.001, mixed-model linear regression).Pathologic retinal lipofuscinosis occurred in very young Grn knockoutmice (1.5 months of age), preceding any other functional or degenerativephenotype in these animals (Fig. 1, C andD, and table S3) (7, 8, 13, 22, 23).Consistent with previous evaluations of the cortex (7), we did not seeincreased retinal lipofuscinosis in aged Grn +/− mice (figs. S1 and S2).

Noninvasive imaging of retinal autofluorescence is routinely per-formed in clinical settingswith confocal scanning laser ophthalmoscopy(cSLO) and can be used to detect retinal lipofuscin deposits. To deter-mine whether retinal lipofuscinosis secondary to PGRN deficiency canbe imaged in vivo, we used a modified cSLO to image the retina of anes-thetized Grn knockout mice. Similar to our observations in fixed tissue,we detected retinal lipofuscin using cSLO inGrn knockoutmice (Fig. 1, Eand F). We simultaneously imaged Grn knockout mice with spectraldomain optical coherence tomography (OCT) to determine whetherindividual lipofuscin deposits could be localized to specific retinal layers.Although most autofluorescent deposits were below the resolution ofOCT in mice, we did note occasional aggregates of subretinal depositsin Grn knockout mice, consistent with our pathological observations(Fig. 1E, insets).

Noninvasive imaging of retinal lipofuscin in heterozygousGRN mutation carriers by autofluorescent retinal imagingWe previously reported retinal thinning in humans with heterozygousGRNmutations (13). We used cSLO to image retinal autofluorescencein 11 heterozygous GRN mutation carriers and 22 age-matched andsex-matched healthy controls (tables S1 and S2). Retinal lipofuscinosis


was seen more often in heterozygous GRN mutation carriers than incontrols (Fig. 2, A to C). Most lipofuscin deposits were not visible onOCT scans through the same region (Fig. 2B). Occasional lipofuscindeposits correlated with subretinal drusen-like aggregates (Fig. 2C).Quantification of subretinal drusen revealed a trend toward increaseddrusen per eye in GRN mutation carriers, but this was not statisticallysignificant (table S2). Heterozygous GRNmutation carriers were abouttwice as likely to have retinal lipofuscin deposits than controls (Fig. 2Dand tables S1 and S3). In addition, both the average number and thearea of lipofuscin deposits were substantially greater in GRNmutationcarriers (Fig. 2, E andF), including thosewhowere clinically asymptomatic(Fig. 2, E and F).

Increased storage material in postmortem cortexof heterozygous GRN mutation carriersWenext asked whether storagematerial deposition also occurred in thefrontal cortex of heterozygous GRN mutation carriers, because degen-eration of this region of the CNS causes many of the clinical symptomsof FTD. Formalin-fixed postmortemhuman cortex samples from15GRNmutation carriers, 16 nondemented control subjects, and 6 Alzheimer’sdisease (AD) subjects were imaged by EM. We noted a marked increasein storage material accumulation in cortical neurons of GRN mutationcarriers but not AD subjects compared to nondemented controls (Fig.2, G and H). Morphologically, the storage material found in the formalin-fixed postmortem cortex fromGRNmutation carriers resembled granularosmiophilic deposits. Such deposits occur in a broad range of NCL sub-types, including recessive infantile-onset forms caused byPPT1mutations(NCL type 1) and an adult-onset form caused by dominant mutations inDNAJC5 (NCL type 4). Both the percentage of neurons that containedstorage material and the number of storage material deposits per neuronwere increased in heterozygous GRNmutation carriers (Fig. 2H). Storagematerial was also present in microglia in heterozygous GRN mutationcarriers (fig. S3). We did not observe an increase in NCL-like storagematerial in postmortem cervical spinal cord tissue from heterozygousGRN mutation carriers, an area of the CNS that does not typicallydegenerate inGRN-related neurodegeneration (fig. S4). Consistent withthe results from EM, we observed an increase in autofluorescent lipo-fuscin deposits in the frontal cortex tissue from GRNmutation carriersby fluorescence microscopy (Fig. 2I). Lipofuscin staining using Sudanblack showed a similar result (Fig. 2, J and K).

NCL-like storage material in lymphoblasts fromheterozygous GRN mutation carriersThe presence of intracellular storage material with characteristic ultra-structural patterns is a defining pathological feature of NCL. Such storagematerial accumulates in neurons and certain non-neuronal cells in NCLpatients. Evaluation of peripheral blood lymphocytes for storagematerialis commonly performed in clinical settings when a diagnosis of NCL isunder consideration. Recently, a patient diagnosed with NCL was post-humously found to carry a heterozygous GRNmutation (c.1477C>TpArg493X), but neither electron micrographs nor tissue samples wereavailable at the time to confirm the original diagnosis (17).We obtainedlymphoblasts that had been banked from this patient and processedthem for EM to evaluate the presence or absence of NCL-like storagematerial. We simultaneously evaluated lymphoblasts from a healthy in-dividual and a patient with juvenile NCL (CLN3 homozygous ~1-kbdeletion). As expected, lymphoblasts carrying a CLN3 mutation hadnumerous enlargedvacuolated structures,many containingmultilamellarfingerprint profile patterns or onion skin-like storage material (Fig. 3A).

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Strikingly, lymphoblasts from the heterozygous GRN mutation carrierhad prominent vacuolated structures containing storage material thatwas morphologically similar to that found in lymphoblasts carrying theCLN3mutation. Storagematerialwas largely absent in lymphoblasts froma healthy control (Fig. 3A, inset)

We next asked whether NCL-like storage material also occurred inheterozygous GRN mutation carriers lacking a clinical diagnosis ofNCL.We cultured lymphoblasts from five heterozygousGRNmutationcarriers and processed them for EM. Siblings with WT GRN alleles ofsimilar age served as controls. Lymphoblasts from theseGRNmutationcarriers hadmembrane-boundmultilamellar NCL-like storagematerialdeposits, similar in morphology to the GRNmutation carrier clinically


diagnosedwithNCL (fig. S5A).When analyzed as a group,we sawmorestorage material in lymphoblasts from GRN mutation carriers than inthose from controls (Fig. 3B). When analyzed as matched sibling pairs,three of theGRNmutation carriers had lymphoblasts withmore storagematerial than their control siblings (fig. S5, B andC).A similar but less pro-nounced difference was observed in the other two pairs (fig. S5, B and C).

Reduced NCL-like storage material in lymphoblastsfrom GRN mutation carriers treated withPGRN-expressing retrovirusWenext determinedwhether theNCL-like storagematerial phenotype inlymphoblasts fromGRNmutation carriers could be rescued by increasing

Fig. 1. Retinal NCL-like storagematerial and lipofuscinosis inGrn knockoutmice. (A) Representative EM images of retinal ganglion cell layer (GCL) neurons from18-month-oldwild-type (WT) andGrn knockout (GrnKO)mice. Note the characteristic fingerprint profile patterns ofNCL-like storagematerial (GrnKO, inset). (B) Representative retinal cross section from24-month-oldGrnKOmice showingautofluorescent lipofuscin throughout the retina. Especially prominent lipofuscinosis occurs in theGCL, innernuclear layer (INL), deep layers of the innerplexiform layer (IPL), outer plexiform layer (OPL), and outer nuclear layer (ONL). Additionally, subretinal drusen-like autofluorescent aggregates occurred inGrn KOmice (white arrow). DAPI,4′,6-diamidino-2-phenylindole; IS, inner segment;OS,outersegment. (C)Representative lipofuscindeposits (redpuncta) in retinalwholemounts fromagingWTandGrnKOmice. (D)Quantificationof lipofuscin deposits from (C) n= 4 to 26mice per age per genotype. ***P < 0.001, one-way analysis of variance (ANOVA) with Tukey’s post hoc test. (E) Representative images of retinallipofuscin in Grn KOmice imaged in vivo using cSLO. Green lines overlaid on cSLO images indicate the location of cross-sectional OCT scans. Occasional subretinal drusen-like depositswere observed inGrnKOmice (insets 1 and2, red arrow), butmost autofluorescent punctawerebelow the limit of detectionofOCT cross-sectional scans through the samearea (insets 3and 4). (F) Quantification of in vivo imaging of retinal lipofuscin from (E). n = 11 to 12 eyes from six mice per genotype. ***P < 0.001, mixed-effect multivariate linear regression model.

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PGRN expression in these cells. A PGRN-expressing retrovirus was usedto transduce lymphoblasts carrying the GRNV200GfsX18 mutation to in-crease PGRN expression to WT levels or higher (Fig. 3, C and D, and


table S3). The NCL-like storage material was reduced in GRNV200GfsX18

lymphoblasts infected with PGRN-expressing retrovirus compared tocontrol cells transduced with an empty retrovirus (Fig. 3, E to G).

Fig. 2. Autofluorescent NCL-likestoragematerial in theCNSofhumanswith heterozygous GRN mutations.(A to C) In vivo imaging of retinal lipo-fuscinosis in humans with GRN haploin-sufficiency. Representative images ofcSLO autofluorescent retinal imaging ofone control subject (Ctrl) and two GRNmutation carriers (GRN mt) are shown.OCTcross-sectional scans (greendashedlines) are shown below the autofluores-cent images. Lipofuscin deposits (insets)occurred more frequently in GRN muta-tion carriers than in control subjects. Mostlipofuscin deposits did not correlatewith detectable abnormalities in OCTcross-sectional scans (B), although somecorrelated with subretinal drusen-likematerial (C, red arrows). Scale bars,1 mm. (D to F) Blinded quantificationof retinal autofluorescence images ob-tained by cSLO. Data are grouped by allGRNmutation subjects versus all controls(n = 11 and 22), presymptomatic GRNmutation subjects versus matchedcontrols (n=8and16), and symptomaticGRN mutation subjects versus matchedcontrols (n = 3 and 6). (D) HeterozygousGRN mutation carriers were more likelythan controls to have retinal lipofuscindeposits; P = 0.027, Fisher’s exact test.Asymptomatic and symptomatic GRNmutation carriers also were more fre-quently found to have retinal lipofuscindeposits, although the difference wasnot significant.P=0.18and0.46, respec-tively, Fisher’s exact test. The number ofaffected subjects is shown inside thebars. *P < 0.05. (E) Quantification of thetotal number of retinal lipofuscin de-posits per subject. Heterozygous GRNmutation carriers had more lipofuscindeposits thandidcontrols, includingsub-groups of asymptomatic and sympto-matic GRN mutation carriers. *P <0.05,***P < 0.001, Mann-Whitney U test.(F) Quantification of the total area ofretinal lipofuscin deposits per subject.
Heterozygous GRNmutation carriers had a greater total area of lipofuscin deposits than did controls, including a subgroup of asymptomatic GRNmutation carriers. **P < 0.01,Mann-Whitney U test. N.S., not significant. (G) Increased accumulation of electron-dense storage material in postmortem cortical tissue from heterozygous GRN mutationcarriers. Shown are representative electron micrographs of formalin-fixed frontal cortex from control and heterozygous GRNmutation carriers. Note the presence of granularosmiophilic deposit–like storagematerial in neurons in cortical tissue fromGRNmutation carriers (insets, yellow arrow), which was rarely observed in control cortical neurons.(H) Quantification of electron-dense storagematerial seen in (G). The percentage of neurons that contained such storagematerial and the number of deposits per neuron areshown. n = 4 to 26 neurons from 15 GRN mutation carriers, 16 control subjects, and 6 AD subjects. Percentage of neurons: ***P < 0.001, one-way ANOVA with Bonferroni’smultiple comparison test; number of deposits per cell: **P < 0.001, mixed model linear regression. (I) Increased autofluorescent lipofuscin aggregates in postmortem corticaltissue from heterozygous GRN mutation carriers. n = 6 to 30 fields of view from nine GRN mutation carriers and nine controls; **P = 0.009, mixed-model linear regression.(J) Representative bright-field images of frontal cortex showing an increase in Sudan black–stained lipofuscin in postmortem cortical tissue from a heterozygous GRN mutationcarrier versus control. (K) Quantification of Sudan black staining seen in (J). The number of lipofuscin deposits per field of view is shown.n=10GRNmutation carriers and 14 controlsubjects; ****P < 0.0001, Student’s t test. Means ± SEM are shown in (E), (F), (H), (I), and (K).
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Fig. 3. GRN haploinsufficiency results in NCL-like storage material in lymphoblasts. (A) NCL-like storage material accumulates in lymphoblasts from a heterozygous GRNmutation carrier who clinically presented with NCL symptoms. Representative images of lymphoblasts from a healthy non–mutation-carrying control subject, an NCL patient(homozygous CLN3mutation carrier), and a heterozygous GRNmutation carrier are shown. Note the similar ultrastructural patterns of the storage material observed in the CLN3mutation carrier and the GRNmutation carrier, consisting of prominent vacuolated structures containing storage material with a fingerprint profile pattern (insets). (B) Quanti-fication of NCL-like storagematerial in lymphoblasts from heterozygousGRNmutation carriers without clinical evidence of NCL versus noncarrier sibling controls. Five paired setsof lymphoblasts from control and heterozygous GRNmutation siblings were imaged with EM, and the number of vacuoles with storagematerial per cell and cross-sectional areaoccupied by storage material containing vacuoles was quantified in a blinded manner. n = 19 to 38 cells per subject from five pairs of GRNmutation carrier and control subjects;***P < 0.001 via mixed-effect multivariate linear regressionmodel. (C toG) Restoring normal PGRN expression in lymphoblasts from heterozygous GRNmutation carriers rescuedthe NCL-like storage material phenotype. (C) Western blot of intracellular PGRN and tubulin showing reduced expression of PGRN in heterozygous GRNExon 8 (IVS7-1g) mutationcarrier lymphoblasts, which was rescued by treatment with a PGRN-expressing retrovirus. Control (empty vector) retrovirus did not alter PGRN expression. GRNExon 8 (IVS7-1g)

mutation carrier lymphoblasts transduced with the PGRN-retrovirus that expressed PGRN at two different levels (endogenous, +; high, ++) were used for EM analysis. (D) Quan-tification of PGRNexpression (normalized to tubulin). n=3wells per group; *P<0.05, **P<0.01, one-wayANOVAwith Sidak’smultiple comparison test. (E) Representative electronmicrographs of heterozygous GRNExon 8 (IVS7-1g) mutation carrier lymphoblasts transduced with control (empty vector) or huPGRN retrovirus expressing PGRN at an endogenouslevel. Lymphoblasts transducedwith PGRN-expressing retrovirus had less storagematerial than did those that were treated with empty vector (insets). (F and G) Quantification ofthe number of vacuoles with storagematerial per lymphoblast (F) or total cross-sectional area of storagematerial per lymphoblast (G) in lymphoblasts transducedwith control orPGRN-expressing retrovirus. n = 45 to 65 cells per group, *P < 0.05, **P < 0.01, one-way ANOVA with Sidak’s multiple comparison test. Means ± SEM are shown (B, D, F, and G).

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Impaired lysosomal function in fibroblasts fromheterozygous GRN mutation carriersIn NCL, lipofuscin accumulation is thought to be the pathological con-sequence of dysfunctional lysosomal proteolysis. We directly assessedlysosomal protease activity in human fibroblasts from heterozygousGRN mutation carriers and control siblings. We measured the activityof cysteine cathepsin proteases in living patient fibroblasts using aquenched fluorescent activity–based probe (24) and found that cathepsinactivity in fibroblasts fromGRNmutation carriers was reduced by ~40%compared to that in fibroblasts from control siblings (Fig. 4, A and B,fig. S6A, and table S3).We also saw reduced cathepsinD activity in fibro-blast lysates from GRNmutation carriers (Fig. 4C and fig. S6B).


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DISCUSSIONOur study shows that FTD caused by PGRN haploinsufficiency sharesseveral fundamental clinicopathological features with NCL. Similar toother areas of the CNS, lipofuscin deposits formed in the retina in Grnknockout mice in a panretinal pattern similar to that seen in humanNCL. Retinal lipofuscin deposition was an early phenotype in thesemice, preceding neuron loss, and could be imaged in vivo. Retinal lipo-fuscin deposits also occurred in humans with heterozygousGRNmuta-tions, including cognitively normal individuals, and could be detectedwith imaging technology used in clinical settings. AutofluorescentNCL-like storage material was present in the frontal cortex of humansubjects with heterozygous GRN mutations. EM analysis of lympho-blasts from heterozygous GRNmutation carriers revealed the presenceofNCL-like storagematerial, regardless ofwhether their clinical syndromehadNCL-like features.Transductionof lymphoblasts fromGRNmutationcarriers with PGRN-expressing retrovirus reduced storage material accu-mulation. Finally, lysosomal protease activity was impaired in fibroblastsfrom heterozygous GRNmutation carriers.

Whereas patients with homozygous GRNmutations have a clinicalsyndrome that closely resembles NCL, those with heterozygous GRNmutations usually present with FTD. Because the primary symptomsand pathological features of FTD and NCL are distinct, homozygousand heterozygousGRNmutationsmay cause neurodegeneration via di-vergent pathophysiological processes (17). In contrast, our study pro-vides direct evidence that core clinicopathological phenotypes of NCLoccur in heterozygous GRN mutation carriers. Vision loss and retinalatrophy are early phenotypes in most patients with NCL. Our recentobservations of moderate retinal thinning in heterozygous GRNmuta-tion carriers, compared to nearly complete vision loss and severe retinalatrophy in homozygousGRNmutation carriers (18), suggest a relation-ship between GRN gene dosage and the severity of retinal involvement.We also found that lipofuscinosis andmembrane-bound storagematerialdeposition—pathologic hallmarks of NCL—occurred in heterozygousGRNmutation carriers, consistent with what has been seen in homozy-gous GRNmutation carriers. A recent finding that an NCL case causedby a homozygousGRNmutation segregated in a familywith neuropatho-logically confirmed FTLD further supports the notion that PGRN-deficient NCL and FTLD are pathophysiologically related (19).

On the basis of our findings, we propose that neurodegeneration isdriven by a common central mechanism in both homozygous and het-erozygousGRNmutation carriers but that the specific clinical syndromeis driven by GRN gene dosage. In homozygous GRNmutation carrierswith complete PGRN loss, disease onset is in early adulthood and NCLsymptoms predominate. In heterozygous GRN mutation carriers withpartial PGRN loss, subclinical NCL features (such as retinal thinning)


are present, but disease onset is in late adulthood and cognitive symptomspredominate. Additional subclinical features could also occur in hetero-zygousGRNmutation carriers that would require further investigation todetect. For example, althoughmotor seizures are not commonly reportedinGRN-associated FTD, prolonged electroencephalographymight revealsubclinical epileptiform activity, similar to that seen in subjects with AD

Fig. 4. Impaired lysosomal protease activity in primary cells from heterozygousGRNmutation carriers. (A) Representative images of fluorescence after staining withBMV109 (red), a cell-permeable probe for active cysteine cathepsin proteases, in HDFsfromheterozygousGRNmutation carrier and control siblings (blue, DAPI nuclear stain).(B) Quantification of BMV109 fluorescence in heterozygous GRNmutation carrier andcontrol HDFs. n = 6wells imaged per HDF line from three pairs ofGRNmutation carrierandmatched sibling controls; ****P<0.0001 viamixed-effectmultivariate linear regres-sion model. AU, arbitrary units. (C) Cathepsin D enzyme activity in HDF lysates fromheterozygous GRN mutation carrier and control sibling pairs. n = 10 assays per HDFline from three pairs of GRN mutation carrier and matched sibling controls; ****P <0.0001 viamixed-effectmultivariate linear regression. Mean enzyme activity, normalizedto matched sibling control lines, ±SEM is shown (B and C).

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(25). Other symptoms seen in homozygousGRN carriers, such as cer-ebellar dysfunction, have not been documented in heterozygous GRNmutation carriers. One possible explanation is that such symptoms canbe notoriously difficult to detect in clinical settings when they are subtleand difficult to localize, for example, behavioral manifestations of cere-bellar dysfunction. Also, potential cerebellar pathology is not always as-sessed in these cases at autopsy. It will therefore be of interest todetermine whether structural changes to the cerebellum occur in theseindividuals, for example, through assessment of cerebellar volume loss orchanges in neural tracts as evidence of disruption of normal cerebellarinput/output. It remains unclear why heterozygous PGRN-deficientmice failed to exhibit NCL-like pathology, despite strikingNCL-like pa-thology in Grn knockout mice. It is possible that mice do not live longenough to develop such pathology or that additional genetic modi-fiers are required forNCL-like pathology to emerge, for example, disease-related TMEM106b polymorphisms.

Notably, we observed NCL-like storage material in a heterozygousGRNmutation carrier who presented with symptoms compatible withlate-stage NCL, for example, limb dystonia, gait abnormalities, andspasticity at the age of 46. Because whole-genome sequencing is morebroadly used to assist in the diagnosis of unusual neurological cases, ad-ditional atypical clinical presentations of heterozygous GRN mutationcarriers may be discovered. Alternatively, this subject may have had ad-ditional partial loss-of-function sequence variants inNCL-related genesthat pushed their clinical syndrome toward an NCL-like phenotype.

PGRN has been reported to regulate a diverse range of cellular func-tions, including cell proliferation, cell survival, tumor invasion, proteinturnover, inflammation, and synaptic plasticity, and more recently,lysosomal function. It has been unclear which of these functions is di-rectly regulated by PGRN, and numerous mechanisms have been pro-posed to explain howpartial PGRN loss causes FTD. PGRN is a secretedglycoprotein and, in neurons, localizes predominantly to endolysosomes(26). It binds directly to sortilin, a transmembrane receptor involved inreceptor scavenging and protein trafficking from the Golgi to the lyso-somes (26). In addition, mutations in the TMEM106b gene markedlyinfluence the age of onset of FTD in heterozygousGRNmutation carriers(27, 28). TMEM106b localizes to endolysosomes and may regulatevesicular transport (29). On the basis of our observations of NCL-likephenotypes in heterozygous GRN mutation carriers, we propose thatendolysosomal dysregulation is a potential centralmechanismunderlyingneurodegeneration in humans with PGRN-deficient FTD.

Because nearly all heterozygousGRNmutations cause FTD throughloss of PGRN expression, it has been speculated that increasing PGRNin GRN mutation carriers using therapeutic interventions should alterdisease progression. However, one significant challenge in the develop-ment of effective therapeutics in FTD has been a lack of markers tomonitor relevant pathophysiological processes. We found that PGRN-deficient lymphoblasts had increasedNCL-like storagematerial and thatincreasingPGRN in these cells could rescue this phenotype, supporting apossible causal role of PGRNdeficiency in endolysosomal dysfunction. Itis largely accepted that the pathological accumulation of lipofuscin inNCL indicates ongoing endolysosomal dysfunction, but future studiesare needed to clarify whether lipofuscin accumulation itself is pathogenic.

Our study further suggests that autofluorescent retinal imaging mayhave potential utility for GRN mutation carriers. Longitudinal auto-fluorescent retinal imaging could be an inexpensive, rapid, and non-invasive means of monitoring lipofuscinosis in the CNS, especiallywhen paired with OCT analysis of retinal tissue loss in GRN mutationcarriers. Future proof-of-concept studies in Grn knockout mice using


serial in vivo retinal imaging could inform whether normalization ofPGRNexpression clears existing lipofuscin, slows future lipofuscin accu-mulation, and prevents retinal neuronal loss. Adaptation of OCT acqui-sition protocols may also permit the detection of abnormalities otherthan the subretinal deposits identified here.

It is important to note the limitations associated with this study.Wefound that PGRN-deficient lymphoblasts had increased NCL-likestorage material and that increasing PGRN in these cells could rescuethis phenotype. However, EM analysis of NCL-like storage material inlymphoblasts is too labor-intensive to be used in the clinic. It remains tobe established whether other assays of impaired lysosome function inPGRN-deficient lymphoblasts could be used to measure disease pro-gression or efficacy of potential treatments. We observed a robustdifference in lipofuscin deposition in the retina and CNS of a single co-hort ofGRNmutation carriers compared to healthy controls. However,given their lack of specificity, these differences would not be sufficientfor identification of presymptomatic individuals without an additionalrisk assessment, that is, genetics or family history. Replication of ourfindings in an independent cohort of GRNmutation carriers would re-inforce our conclusions.

MATERIALS AND METHODSStudy designThe overall aim of the study was to determine whether cells and tissuefrom heterozygous GRNmutation carriers exhibited evidence of NCL-like accumulation of lysosomal storage material and lysosomal dys-function. The objective of the first portion of the studywas to determinewhether NCL-like storage material occurred in Grn knockout miceusing EM, histology, and live imaging techniques. The objective of thesecond portion of the study was to determine whether NCL-like storagematerial was present in the CNS of heterozygousGRNmutation carriersusing retinal imaging of living subjects and analysis of postmortem hu-man cortical tissue by EM. Subjects with heterozygous GRNmutationsand age-matched and sex-matched control subjects without knownneurological diseases were enrolled in the study through the Universityof California, San Francisco (UCSF)Memory andAging Center in 2011–2014 as part of a larger study to evaluate retinal manifestations of neuro-degenerative diseases. Predefined exclusion criteria included a history ofophthalmologic disease, including glaucoma, age-related macular de-generation (AMD), diabetic retinopathy, and ocular surgery (except cata-ract surgery).OneGRNmutation carrier hadAMD(dry) andwas excludedfrom the analysis; the remainder did not have any of the above exclusioncriteria. GRN mutation carriers also underwent a standardized neuro-logical evaluation by a board-certified neurologist with fellowshiptraining in behavioral neurology. Clinical disease rating (CDR) scoreswere assigned to each subject on the basis of the result of this evaluation.For the purposes of this study, subjects were classified as asymptomatic(CDR = 0) or symptomatic (CDR > 0). Written informed consent wasobtained from all participants with capacity. In those unable to provideinformed consent because of diminished capacity, assent was obtainedfrom the participant andwritten consent was obtained from a designatedsurrogate decision-maker. The study protocol was approved by theUCSFCommittee on Human Research [IRB (Institutional Review Board) #11-05333]. The objective of the third portion of the study was to evaluateprimary cells from heterozygous GRN mutation carriers for evidence oflysosomal dysfunction using EM analysis of storage material accumula-tion and functional readouts of lysosomalhydrolase activity.All datawereincluded (there was no outlier exclusion).

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Lymphoblast analysisEpstein-Barr virus–transformed lymphocytes from deidentified humansubjects were grown in RPMI + 10% fetal calf serum + 1% penicillin/streptomycin. Equal numbers of cells from each line were seeded into10ml of medium in T75 tissue culture flasks, andmediumwas doubledin volume each time cells reached saturation (determined by mediumpH)until the total volumewas 40ml. Cellswere again allowed to expanduntil saturation density and were then pelleted and gently resuspendedin ice-cold EM fixative [2.5% glutaraldehyde, 2% paraformaldehyde,and 0.025% calcium chloride, in a 0.1 M sodium cacodylate buffer(pH7.4)]. After a 15-min incubation in fixative on ice, cellswere repelletedat 5000 rpm in a swinging-bucket rotor centrifuge for 10 min at 4°C.The fixative was removed, and fresh fixative was gently layered ontop of the cell pellet. Samples were centrifuged again at 5000 rpm for10 min at 4°C and then processed further for EM.

Retroviral transduction of lymphoblastsUntagged human progranulin was cloned into a murine stem cell virus(MSCV) backbone vector that coexpressed mRuby as a fluorescentmarker, with an empty vector serving as a control. 293T cells were co-transfected with the MSCV vector and pCL-Ampho via Lipofectamine2000, and viral supernatant was collected 2 days later and filteredthrough a 0.45-mm filter.GRNmutation lymphoblasts were resuspendedin empty vector or PGRN-expressing retroviral supernatant andmonitored for expression via fluorescencemicroscopy. PGRN-expressingcells were sorted into two groups by fluorescence-activated cell sorting:“high PGRN-expressing” cells were the brightest 5% of cells sorted,and “low PGRN-expressing” cells were the remaining mRuby-positivecells. These groups were then expanded in parallel as described aboveand processed for EM.

Electron microscopyLymphoblast pellets were generated as described above, mouse retinaltissue was fixed directly after harvest in EM fixative. Formalin-fixedpostmortem human cortex was postfixed in EM fixative before furtherprocessing. For lymphoblasts and retinal tissue, subsequent processingwas performed in a Leica Lynx automatic tissue processor. Briefly, thetissue was postfixed in osmium tetroxide, stained en bloc with uranylacetate, dehydrated in graded ethanol solutions, infiltratedwithpropyleneoxide/Epon mixtures, embedded in pure Epon, and polymerizedovernight at 60°C. One-micrometer sections were cut, stained with tolui-dine blue, and examined by light microscopy. Representative areas werechosen for electronmicroscopic study, and the Epon blocks were trimmedaccordingly. Thin sectionswere cutwith an LKB 8801 ultramicrotome anddiamondknife, stainedwith lead citrate, and examined in anFEIMorgagnitransmission electron microscope.

Quantification of lymphoblast storage materialEM images were blindly acquired by an experienced EM technologist,who, to reduce the chance of bias, was instructed to takewhole-cell imagesat ×1200 magnification of the first 20 to 30 cells encountered during im-aging, in which the nucleus was bisected at the plane of section. Blindedquantification of lymphocyte storage material was then carried out. Thetotal number of vacuoles per lymphocyte with NCL-like storage material(defined by the presence of multilamellar, fingerprint profile storagematerial or the granular osmiophilic deposit profile storage material)was determined. Simultaneously, regions of interest around each vacuolecontaining storage material were traced, and the summed area of storagematerial–containing vacuoles per lymphocyte was determined.


Quantification of cathepsin activity (fluorescent-based andcathepsin D activity assay)Human-derived fibroblasts (HDFs) were passaged into 10- or 15-cmtissue culture–treated culture dishes, allowed to expand to confluency,and cultured in 10% fetal bovine serum (FBS)/Dulbecco’s modifiedEagle’smedium (DMEM) for 1month. For experiments, 0.25% trypsin-EDTA solution was used to dissociate cells, and the cells were plated in100-ml aliquots to a density of 2000 cells per well in TPP 96-well tissueculture plates. After adhesion of cells for 24 hours, the medium wasremoved from wells, and a probe, BMV109, was added to each well(0.25 mMBMV109 in 100 ml of FBS/DMEM). The probe was incubatedwith cells for 2 hours, after which point the probe-containing mediumwas removed, andwells werewashedwith nonfluorescentDMEM threetimes. Cells were incubated with Hoechst stain (1.6 mM) in nonfluores-cent DMEM. The plates were imaged at ×10magnification on CellomicsArrayScan, with an exposure of 1 s. Two channels were used for imaging:386 nm for nuclear stain and 650 nm for BMV109. The signal was quan-tified after imaging with Cellomics software using Cell Health Profiling.Wells with no probe were used as blanks. For cathepsin D activity assays,a fluorometric cathepsin D assay kit was used (BioVision). Briefly, HDFswere lysed with CD lysis buffer, incubated on ice for 10min, and clarifiedvia centrifugation at 16,000g for 5 min, and total protein levels were nor-malized across samples. Lysates were added to the master assay mix,mixed, and incubated for 1 hour at 37°C. Fluorescence was read on a flu-orescent plate reader (excitation, 328 nm; emission, 460 nm).

In vivo retinal imaging in miceAll animals were maintained in full-barrier facilities free of specificpathogens on a 12-hour light/dark cycle with food andwater ad libitum.Experimentswere conducted in compliancewith theARVO(Associationfor Research in Vision and Ophthalmology) Statement for the Use ofAnimals in Ophthalmic and Visual Research, and all protocols wereapproved by the Institutional Animal Care and Use Committee at theUniversity ofCalifornia, SanFrancisco (#AN087501-02A).Grn knockoutmice were fully backcrossed into C57BL/6J, and WT mice of the samegenetic background served as controls. Mice were anesthetized with asteady flow of 1.5 to 3% isofluorane, eyes were anesthetized with propa-caine, dilated with 1% tropicamide and 2.5% phenylephrine, and corneaswere keptmoist with 2.5%methylcellulose. A contact lenswas placed overthe mouse eye to improve the optics of the system. Retinal images weretaken with a modified confocal scanning laser ophthalmoscope/OCTdevice (Heidelberg Spectralis, Heidelberg Engineering). After assuringcentered beam placement by infrared cSLO and 90° position of the retinato the beam in the vertical and horizontal planes byOCT, autofluorescentretinal images were then taken at 457-nm excitation and 500-nm long-pass filter detection using TruTrack eye-tracking software, averagingat least 80 frames for each image, followed by OCT volume scans (100automatic real-time, high resolution) through selected areas of interest.

Retinal lipofuscin quantification in miceEyes were enucleated and fixed in 4% paraformaldehyde overnight.Retinas were dissected out and mounted between slides and coverslipsin a whole-mount configuration. Five separate fields of view, equallyspaced around the perimeter of the retina, were imaged in each retina,located about 500 mmfrom the peripheral edge of the retina. Full-thicknessconfocal images were obtained at 2-mm step sizes using a Nikon SpinningDisk confocal microscope in the green fluorescent protein channel toimage autofluorescent lipofuscin deposits. Individual lipofuscin de-posits weremarkedwith the cell counter feature by a blinded technician,

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and the total number of lipofuscin deposits per z-stack was quantified.The average number of lipofuscin deposits per z-stack across all fivefields of view was determined for each mouse.

Human retinal imagingAfter pupillary dilation with 1% tropicamide ophthalmic solutions(Akorn), study participants underwent retinal autofluorescence imagingwith a cSLO and OCT (Heidelberg Spectralis). cSLO imaging was per-formed using the standard autofluorescence settings of the device usingTruTrack eye-tracking software, averaging at least 80 frames for eachimage. Trained technologists who were blinded to the mutation statusacquired the images using standardized illumination and gain settings.Image qualitywas assessed by a blinded neuro-ophthalmologist, and onlyeyes with in-focus retinal images of sufficient quality to assess for the pres-ence or absence of lipofuscin were analyzed further. Blinded quantifica-tion of retinal lipofuscin was performed jointly by a neuro-ophthalmologist(A.J.G.) and behavioral neurologist (M.E.W.). For each eye, the number ofindividual lipofuscin deposits was counted, and regions of interest weredrawn around each lipofuscin deposit. The total number of lipofuscindeposits and the summed area of lipofuscin deposits were then cal-culated for each eye.

Quantification of lipofuscin in postmortem humancortical tissueFormalin-fixed, paraffin-embedded, deidentified postmortem humanfrontal cortex and cervical spinal cord from heterozygousGRNmutationcarriers, AD subjects, and age-similar and sex-similar control subjectswithout a clinical or pathological diagnosis of neurodegenerative diseasewere obtained from theUniversity of BritishColumbia, theUCSFNeuro-degenerative Disease Brain Bank, and Northwestern University. Thework was deemed not human-subjects research per UCSF CommitteeonHumanResearch guidelines. 7.5-mm-thick sectionswere cut, dewaxed,and counterstained with DAPI (4′,6-diamidino-2-phenylindole). Auto-fluorescence imaging of lipofuscin and imaging of Sudan black–stainedsections was performed by a blinded technician using a 20× objectiveand Cy5 filter set and bright field. Six to 30 nonoverlapping fields ofview of superficial cortex were imaged, and regions of interest weredrawn around lipofuscin deposits via an automated algorithm (Volocitysoftware). The total area of cortex occupiedby lipofuscindepositswas thencalculated for each field of view. For EM analysis of storage material frompostmortem human cortex, formalin-fixed cortex was post-fixed in EMfixative and processed for EM analysis, as above. Quantification of thepercentage of neurons with lysosomal storage deposits and number oflysosomal storage deposits per cell was performed blindly.

StatisticsWe used unpaired two-tailed t tests for normally distributed data andMann-WhitneyU tests for nonparametric data in experiments inwhichtwo comparison groups were involved. In experiments in which morethan two comparison groups were involved, we used an ANOVA testwith post hoc testing. A mixed-effects multivariable linear regressionanalysis, when used, accounted for intramouse and intraindividual var-iability. Statistical testing was performed using Stata 12.0 and Prism 6.

SUPPLEMENTARY MATERIALSwww.sciencetranslationalmedicine.org/cgi/content/full/9/385/eaah5642/DC1Fig. S1. Increased lipofuscin in retinas from aged Grn−/− but not Grn+/− mice.Fig. S2. Increased subunit C deposition in retinas of Grn−/− mice.Fig. S3. Progranulin-deficient microglia accumulate NCL-like lysosomal storage material.


Fig. S4. EM analysis of postmortem spinal cord from control subjects and heterozygous GRNmutation carriers.Fig. S5. Increased NCL-like storage material in lymphoblasts from heterozygous GRN mutationcarriers versus sibling controls.Fig. S6. Reduced cysteine cathepsin protease activity and cathepsin D activity in HDFs fromheterozygous GRN mutation carriers versus sibling controls.Table S1. Patient characteristics and quantification of human retinal autofluorescence imaging.Table S2. Patient characteristics and quantification of human OCT macular cross-sectionalscans.Table S3. Data values for small sample sizes.

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Acknowledgments: We thank J. Herz, L. Mitic, and R. Pearlman for their helpful feedbackand A. Karydas for assistance in genetic testing of study participants and for help inobtaining lymphoblastoid cell lines. We thank the Program in Membrane BiologyMicroscopy Core at Massachusetts General Hospital and A. Simas for technicalassistance in some of the lymphoblastoid EM studies. S. Arnow assisted in humanretinal imaging. Funding: This study was funded by NIH (K08EY023610), BluefieldFoundation, American Brain Foundation (to M.E.W.); That Man May See (AW141505and AW121324), Research to Prevent Blindness (Core), and National Eye Institute(PAR 13-269) (to A.J.G.); NIH (R01AG036884 and R01AG051390) and Consortium forFrontotemporal Dementia Research (to L.G.); NIH grant R01 NS098516, VA Merit AwardsI01 RX002133 and I01 BX001108, and Consortium for Frontotemporal DementiaResearch (to E.J.H.); NIH: National Institute of Neurological Disorders and Stroke(R01NS073813) (to S.L.C.); Alzheimer’s Disease Center P30 A.13854 (to E.B. and M.-M.M.)and P30 AG13854 (to M.-M.M.); Philippe Foundation (to H.B.); National MultipleSclerosis Society (FG 20102-A-1) (to A.C.-H.); and Consortium for Frontotemporal DementiaResearch and NIH grants AG023501 and AG19724 (to W.W.S.). Author contributions:Experimental design: M.E.W., R.C., M.T., J.M.G., S.L.C., L.G., and A.J.G. Collectionof data: M.E.W., R.C., H.-Y.H., C.L., A.T., C.C., S.L., H.B., S.S.M., P.A., A.C.-H., M.V.A., and J.F.S.Data analysis and interpretation: M.E.W., R.C., H.-Y.H., C.C., S.L., M.T., A.T., H.B., M.R., J.M.G.,A.C.-H., M.V.A., W.W.S., B.L.M., J.F.S., E.J.H., L.G., and A.J.G. Subject recruitment: R.C.,A.T., and W.W.S. Provision of reagents: W.W.S., I.R.M., J.F.S., E.B., M.-M.M., M.S.B., S.L.C., andE.J.H. Writing of the manuscript: M.E.W., L.G., and A.J.G. Competing interests: L.G. hasreceived honoraria from Sanford Burnham Prebys Medical Discovery Institute. W.W.S.has received consulting fees from Merck Inc. J.M.G. has received consulting fees fromGenentech and MedImmune, medical legal consulting, and research support fromGenentech, Quest Diagnostics, and MedDay. P.A. has received speaker honoraria,consultation fees, or travel support from Allergan, Biogen, Eisai, GlaxoSmithKline, Ipsen,Merz, Novartis, and Teva and research grants from Biogen, Ipsen, Merz, Novartis, andRoche. B.L.M. serves as the medical director for the John Douglas French Alzheimer’sFoundation; the scientific director for the Tau Consortium; the director/medical advisoryboard member of the Larry L. Hillblom Foundation; a scientific advisory board memberfor the National Institute for Health Research Cambridge Biomedical Research Centre andits subunit, the Biomedical Research Unit in Dementia (U.K.); and a board member forthe American Brain Foundation. A.J.G. is a founder of Inception 5 Biosciences and is onthe scientific advisory board of Bionure. All other authors declare that they have nocompeting interests.

Submitted 8 October 2014Resubmitted 14 July 2016Accepted 21 November 2016Published 12 April 201710.1126/scitranslmed.aah5642

Citation: M. E. Ward, R. Chen, H.-Y. Huang, C. Ludwig, M. Telpoukhovskaia, A. Taubes,H. Boudin, S. S. Minami, M. Reichert, P. Albrecht, J. M. Gelfand, A. Cruz-Herranz, C. Cordano,M. V. Alavi, S. Leslie, W. W. Seeley, B. L. Miller, E. Bigio, M.-M. Mesulam, M. S. Bogyo,I. R. Mackenzie, J. F. Staropoli, S. L. Cotman, E. J. Huang, L. Gan, A. J. Green, Individuals withprogranulin haploinsufficiency exhibit features of neuronal ceroid lipofuscinosis. Sci. Transl.Med. 9, eaah5642 (2017).

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lipofuscinosisIndividuals with progranulin haploinsufficiency exhibit features of neuronal ceroid

Bogyo, Ian R. Mackenzie, John F. Staropoli, Susan L. Cotman, Eric J. Huang, Li Gan and Ari J. GreenMarcel V. Alavi, Shannon Leslie, William W. Seeley, Bruce L. Miller, Eileen Bigio, Marek-Marsel Mesulam, Matthew S.Sakura S. Minami, Meredith Reichert, Philipp Albrecht, Jeffrey M. Gelfand, Andres Cruz-Herranz, Christian Cordano, Michael E. Ward, Robert Chen, Hsin-Yi Huang, Connor Ludwig, Maria Telpoukhovskaia, Ali Taubes, Helene Boudin,

DOI: 10.1126/scitranslmed.aah5642, eaah5642.9Sci Transl Med

-associated FTD and NCL.GRNcentral mechanism in both asystem, and their cells exhibit signs of lysosomal dysfunction. These findings implicate lysosomal dysfunction as

mutations accumulate storage material throughout the central nervousGRNindividuals with heterozygous mutations exhibit clinical and pathological features that are strikingly similar to NCL. Like NCL patients,GRN

. show that patients with heterozygous et alprogranulin-deficient FTD and NCL remains unexplored. Now, Ward (NCL), a lysosomal storage disease that mainly affects children. The underlying relationship between

mutations cause neuronal ceroid lipofuscinosisGRN(FTD) in the elderly population, whereas homozygous mutations lead to progranulin haploinsufficiency and cause frontotemporal dementiaGRNHeterozygous

Connecting the dots in neurodegenerative disease

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neurodegenerative disease individuals with progranulin ...william w. seeley,3 bruce l. miller,3...

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